Uv visible spectrophotometer

The extracts were solubilized in methanol (20 mg·mL⁻¹), followed by dilution in distilled water. Then, an aliquot of 500 μL of extract was added to 250 μL of Folin–Ciocalteu reagent (1 N) and 1250 μL of sodium carbonate (75 g·L⁻¹). The reaction was kept in the dark and after 30 min, the absorbance of the mixture was read at 760 nm using a UV-visible spectrophotometer (Amersham Biosciences, Little Chalfont, UK). The experimental calibration curve was prepared using gallic acid (Sigma Aldrich, St. Louis, MO, USA) at concentrations of 0.5 to 10.0 mg·L⁻¹, and the content of total phenolics was expressed as gallic acid equivalents (GAE) in milligrams per gram of extract (mg GAE·g⁻¹).

qRT-PCR was performed according to the manufacturer's instructions (TaKaRa, Japan). Briefly, total RNA was extracted from cells using RNAiso Plus (Takara, Japan), after which the RNA concentration and quality was determined using a UV/Visible spectrophotometer (AmershamBiosciences, Sweden) to measure absorbance at 260 nm and 280 nm. The RNA was then reverse transcribed to cDNA synthesis using a PrimeScript RT reagent Kit according to the manufacturers (Takara, Japan) instructions. For qRT-PCR, the primers for PER1, KI-67, MDM2, C-MYC, p53, BAX, BCL-2, MMP2, MMP9, TIMP-2 and VEGF were designed using Oligo17.0 software and are listed in Table 3. β-actin served as a normalization control. The reaction mixture for qPCR contained 12.5 μl of 2×SYBR Premix Ex TaqTMII, 2 μl of cDNA template, 1μl of 0.4 μM forward and reverse primers and double distilled H₂O in a total volume of 25 μl. qPCR was performed using a C-1000TM Thermal Cycler (Bio-Rad, USA). The PCR protocol entailed 1 cycle at 95°C for 1.5 min and 40 cycles of 10 s at 95°C and 30s at 60°C. The cycle threshold (Ct) values were determined and normalized against the expression of β-actin in each sample, and the data were analyzed using the2^−ΔΔCt method. The assays were done in triplicate.

Methods described in our previous report [35 (link)] were also used in performing total RNA preparation and cDNA synthesis. Monophasic solution of phenol and guanidine isothiocyanate (TRIzol Reagent, Invitrogen, Carlsbad, CA, USA) were used for the extraction of total RNA. RNA concentration was determined by a UV-visible spectrophotometer (Amersham Biosciences, Piscataway, NJ, USA) at 260 nm. Aliquots (1.0 µg) of RNA from each sample were reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (MML-V RTase, Promega). 1× RT-buffer, 2 mM deoxynucleotide triphosphates (dNTPs, Promega), 0.2 pM oligo dT primer (16-mer) (Bioneer Inc., Daejeon, Korea), and MML-V RTase (2.5 units/µL) in 20 µL reaction volumes were added in performing reverse transcription. Then, the samples were incubated at 42 °C for 60 min and then stored at −20 °C.